Homologous recombination

What is homologous recombination?

The breakage and rejoining of DNA molecules using regions of homology to form new combinations.

Main biological roles of homologous recombination

Generate genetic diversity through allele exchange during meiosis; repair double-stranded DNA breaks.

Difference between mitotic and meiotic recombination

Meiotic recombination creates new allele combinations; mitotic recombination primarily repairs DNA damage in sister chromatids.

Why homologous recombination increases genetic diversity

Crossing-over shuffles alleles between homologous chromosomes during meiosis.

General features of homologous recombination

Requires homologous chromosome pairs; occurs randomly; exchanges DNA without altering sequence; forms heteroduplex joints.

What is a heteroduplex joint?

A region where one strand from each of two homologous DNA molecules base-pairs with each other.

Two major pathways of homologous recombination

Single-strand break (SSB) model and double-strand break (DSB) model.

Shared feature of both HR pathways

Formation of Holliday junction intermediates.

Basic steps of homologous recombination

Alignment → Strand exchange → Holliday junction formation → Branch migration → Resolution.

Definition of branch migration

Movement of the Holliday junction along the DNA through repeated melting and reformation of base pairs.

Outcome of Holliday junction resolution

Two recombinant DNA molecules, with either crossover or non-crossover products.

What initiates the single-strand break model?

Nicks (single breaks) introduced in each DNA duplex.

Key feature of the SSB model

Strand invasion following peeling away of strands at the nick site.

What initiates the double-strand break (DSB) model?

Double-stranded break formation followed by resection.

Steps unique to the DSB model

Resection of 5' ends → Strand invasion → D-loop formation → Second end capture → Two Holliday junctions.

Name the major proteins in E. coli homologous recombination

RecBCD, RecA, RuvA, RuvB, DNA polymerase/ligase, RuvC.

Main function of RecBCD

Resection of DNA ends; helicase, exonuclease, endonuclease, and ATPase activities.

What are Chi sites?

8-bp DNA sequences (5’-GCTGGTGG-3’) that enhance recombination and modulate RecBCD activity.

Effect of Chi sites on RecBCD

Stimulate RecBCD to generate a long 3’ single-stranded overhang for RecA loading.

Function of RecA

Catalyzes strand invasion and strand exchange; binds strongly to ssDNA; ATPase.

How RecA catalyzes strand exchange

RecA-ssDNA filament aligns with homologous dsDNA, then exchanges strands, displacing one strand.

Role of RuvA

Holliday junction–specific DNA-binding protein; recruits RuvB.

Role of RuvB

Helicase that drives branch migration using ATP.

Role of RuvC

Resolvase that nicks two strands at the Holliday junction to separate recombinant DNA molecules.

What is the outcome after RuvC cleavage?

Resolution of the Holliday junction into separate recombinant DNA duplexes.

Practical uses of recombination

Mapping gene distances using recombination frequency; constructing gene knockout organisms.

Why recombination frequency reflects gene distance

Genes farther apart experience more crossovers than genes close together.

What is a D-loop?

A displacement loop formed during strand invasion where ssDNA pairs with the complementary strand of a duplex.

Why HR is highly accurate

It exchanges DNA only between homologous sequences without altering nucleotide sequence.

Primary benefit of HR during mitosis

Repair of damaged DNA, especially double-strand breaks.

Number of Holliday junctions in the DSB model

Two Holliday junctions are formed.

What drives formation of ssDNA in the DSB model?

RecBCD-mediated helicase and nuclease activity.

RecA preference for DNA types

Binds ssDNA strongly; binds dsDNA weakly.

Why ATP is needed by RecA

ATP hydrolysis drives strand invasion and exchange steps.

What does branch migration accomplish?

Extends heteroduplex regions and moves the crossover point.

What ensures correct pairing in strand invasion?

Homology recognition between ssDNA and the dsDNA target.

Why are Chi sites considered recombination hotspots?

They increase recombination frequency by modifying RecBCD activity.

What happens after strand extension in the DSB model?

Gap filling by DNA polymerase and nick sealing by ligase.

What is gene mapping by recombination?

Using crossover frequency to determine physical distances between genes.